Evaluation of Uracil, Sodium Ascorbate, and Rosiglitazone as Promoters of Urinary Bladder Transitional Cell Carcinomas in Male Sprague-Dawley Rats.

The purpose of this study was to establish a 2-stage model of urinary bladder carcinogenesis in male Sprague-Dawley rats to identify tumor promoters. In phase 1 of the study, rats ( n = 170) were administered 100 mg/kg of the tumor initiator, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), twice weekly by oral gavage (po) for a period of 6 weeks. Phase 2 consisted of dividing rats into 4 groups ( n = 40 per group) and administering one of the following for 26 weeks to identify putative tumor promoters: (1) vehicle po, (2) 25 mg/kg/day rosiglitazone po, (3) 5% dietary sodium l-ascorbate, and (4) 3% dietary uracil. Rats were necropsied after 7.5 months, and urinary bladders were evaluated by histopathology. BBN/vehicle treatments induced the development of urothelial hyperplasia (83%) and papillomas (15%) but no transitional cell carcinomas (TCCs). Rosiglitazone increased the incidence and severity of papillomas (93%) and resulted in TCC in 10% of treated rats. Uracil was the most effective tumor promoter in our study and increased the incidence of papillomas (90%) and TCC (74%). Sodium ascorbate decreased the incidence of urothelial hyperplasia (63%) and did not increase the incidence of urothelial papillomas or TCC. These data confirm the capacity of our 2-stage model to identify urinary bladder tumor promoters.

The synthesis of [14 C]4-acetylphenylalanine, effect on cell viability, and assessment of protein incorporation in male rat hepatocytes.

PEGylation is a proven approach to prolonging the duration of action and enhancing biophysical solubility and stability of peptides. 4-Acetylphenylalanine is a novel amino acid with a ketone side chain that is uniquely reactive in proteins. The ketone functionality can react with an aminooxy functionalized polyethyleneglycol polymer to form a stable oxime adduct of the protein. One concern with using unnatural amino acids, such as 4-acetylphenylalanine, is the possibility of it being cleaved from the peptide and becoming incorporated into endogenous proteins. To determine whether this occurs, an in vitro experiment to assess the cell viability and amino acid incorporation into endogenous proteins using primary male rat hepatocytes in the presence of [14 C]4-acetylphenylalanine, 4 or [14 C(U)]L-phenylalanine was conducted. [14 C]4-acetylphenylalanine, 4 was prepared in 2 radiochemical steps from [1-14 C]acetyl chloride in an overall 8% radiochemical yield and in 99.9% radiochemical purity. The results showed that there was no evidence of carbon-14 incorporation into hepatocyte endogenous proteins with [14 C]pAcF and there was no difference between it and L-phenylalanine in cell viability assessments at any of the concentrations studied between 0.1 and 1000 µM.

Evaluation of microRNAs-208 and 133a/b as differential biomarkers of acute cardiac and skeletal muscle toxicity in rats.

Conventional circulating biomarkers of cardiac and skeletal muscle (SKM) toxicity lack specificity and/or have a short half-life. MicroRNAs (miRNAs) are currently being assessed as biomarkers of tissue injury based on their long half-life in blood and selective expression in certain tissues. To assess the utility of miRNAs as biomarkers of cardiac and SKM injury, male Sprague-Dawley rats received a single dose of isoproterenol (ISO); metaproterenol (MET); allylamine (AAM); mitoxantrone (MIT); acetaminophen (APAP) or vehicle. Blood and tissues were collected from rats in each group at 4, 24 and 48h. ISO, MET, and AAM induced cardiac and SKM lesions and APAP induced liver specific lesions. There was no evidence of tissue injury with MIT by histopathology. Serum levels of candidate miRNAs were compared to conventional serum biomarkers of SKM/cardiac toxicity. Increases in heart specific miR-208 only occurred in rats with cardiac lesions alone and were increased for a longer duration than cardiac troponin and FABP3 (cardiac biomarkers). ISO, MET and AAM induced increases in MyL3 and skeletal muscle troponin (sTnl) (SKM biomarkers). MIT induced large increases in sTnl indicative of SKM toxicity, but sTnl levels were also increased in APAP-treated rats that lacked SKM toxicity. Serum levels of miR-133a/b (enriched in cardiac and SKM) increased following ISO, MET, AAM and MIT treatments but were absent in APAP-treated rats. Our results suggest that miR-133a/b are sensitive and specific markers of SKM and cardiac toxicity and that miR-208 used in combination with miR-133a/b can be used to differentiate cardiac from SKM toxicity.

Utilization of the Zucker Diabetic Fatty (ZDF) Rat Model for Investigating Hypoglycemia-related Toxicities.

Glucokinase (GK) catalyzes the initial step in glycolysis and is a key regulator of glucose homeostasis. Therefore, glucokinase activators (GKa) have potential benefit in treating type 2 diabetes. Administration of a Bristol-Myers Squibb GKa (BMS-820132) to healthy euglycemic Sprague-Dawley (SD) rats and beagle dogs in 1 mo toxicology studies resulted in marked and extended hypoglycemia with associated clinical signs of toxicity and degenerative histopathological changes in the stomach, sciatic nerve, myocardium, and skeletal muscles at exposures comparable to those expected at therapeutic clinical exposures. To investigate whether these adverse effects were secondary to exaggerated pharmacology (prolonged hypoglycemia), BMS-820132 was administered daily to male Zucker diabetic fatty (ZDF) rats for 1 mo. ZDF rats are markedly hyperglycemic and insulin resistant. BMS-820132 did not induce hypoglycemia, clinical signs of hypoglycemia, or any of the histopathologic adverse effects observed in the 1 mo toxicology studies at exposures that exceeded those observed in SD rats and dogs. This indicates that the toxicity observed in euglycemic animals was secondary to the exaggerated pharmacology of potent GK activation. This study indicates that ZDF rats, with conventional toxicity studies, are a useful disease model for testing antidiabetic agents and determining toxicities that are independent of prolonged hypoglycemia.

Nonclinical toxicology assessments support the chronic safety of dapagliflozin, a first-in-class sodium-glucose cotransporter 2 inhibitor.

Dapagliflozin, a first-in-class, selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), promotes urinary glucose excretion to reduce hyperglycemia for the treatment of type 2 diabetes. A series of nonclinical studies were undertaken to evaluate dapagliflozin in species where it was shown to have pharmacologic activity comparable with that in humans at doses that resulted in supratherapeutic exposures. In vitro screening (>300 targets; 10 µmol/L) indicated no significant off-target activities for dapagliflozin or its primary human metabolite. Once daily, orally administered dapagliflozin was evaluated in Sprague-Dawley rats (≤6 months) and in beagle dogs (≤1 year) at exposures >5000-fold those observed at the maximum recommended human clinical dose (MRHD; 10 mg). Anticipated, pharmacologically mediated effects of glucosuria, osmotic diuresis, and mild electrolyte loss were observed, but there were no adverse effects at clinically relevant exposures, including in the kidneys or urogenital tract. The SGLT2-/- mice, which show chronic glucosuria, and dapagliflozin-treated, wild-type mice exhibited similar safety profiles. In rats but not dogs, dapagliflozin at >2000-fold MRHD exposures resulted in tissue mineralization and trabecular bone accretion. Investigative studies suggested that the effect was not relevant to human safety, since it was partially related to off-target inhibition of SGLT1, which was observed only at high doses of dapagliflozin and resulted in intestinal glucose malabsorption and increased intestinal calcium absorption. The rigorous assessment of supra- and off-target dapagliflozin pharmacology in nonclinical species allowed for a thorough evaluation of potential toxicity, providing us with confidence in its safety in patients with diabetes.

Serum microRNAs-217 and -375 as biomarkers of acute pancreatic injury in rats.

Pancreatic injury in rats is primarily detected through histopathological changes and conventional serum biomarkers such as amylase and lipase. However, amylase and lipase have a short half-life and are markers of acinar, not islet cell injury. We investigated whether circulating microRNA (miR) levels that are enriched in acinar cells (miR-217, miR-216a/b) or islet cells (miR-375) could serve as markers of pancreatic injury. Rats were treated with a single dose of either vehicle, streptozotocin (STZ), caerulein, or acetaminophen (APAP), and necropsied at 4, 24, and 48h. Pancreas, liver, heart, kidney and skeletal muscle were analyzed for histopathology. Blood was collected at necropsy and processed to serum for amylase/lipase enzymatic determinations and miR qPCR analysis. Caerulein induced degeneration/necrosis of acinar cells at 4h that persisted for 48h. Caerulein-induced injury was associated with increases in serum amylase/lipase (4h), miR-216a/b (4, 24h). In contrast, serum miR-217 was detected at all time points examined. STZ did not induce increases in either amylase or lipase but did induce increases in miR-375 levels at 4 and 24h. No increases in miR-375 were observed in caerulein-treated rats, and no increases were observed in miR-217 and miR-216a/b in STZ-treated rats. APAP induced centrilobular necrosis in the liver 24h after treatment, but did not induce pancreatic injury or increases in miR-217 or miR-375. Our results suggest that miR-217 and miR-375 represent promising biomarkers of pancreatic injury in rats.

Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells were incubated with 6-hydroxydopamine (6-OHDA) for 4 and 24 h to examine the mechanism of cell death and to determine the time-dependent effects of 6-OHDA on cellular glutathione status. After 4 h, 6-OHDA significantly depleted cellular ATP and GSH concentrations with only slight increases in cell death. GSH:GSSG ratios and mitochondrial membrane potential (Deltapsim) were significantly decreased during 4 h incubations with 6-OHDA. High concentrations of 6-OHDA (100 microM) induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells within 4 h leading to cell death. In 24 h incubations, 25 and 50 microM 6-OHDA significantly decreased ATP concentrations; however, significant increases in cell death were only observed with 50 microM 6-OHDA. 6-OHDA induced a concentration-dependent increase in GSH and total glutathione concentrations after 24 h. After exposure to 50 microM 6-OHDA, GSH concentrations were increased up to 12-fold after 24 h with no change in the GSH:GSSG ratio. Gene analysis suggests that the increase in GSH concentration was due to increased expression of the GSH synthesis genes glutamate cysteine ligase modifier and catalytic subunits. Our results suggest that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular GSH concentrations.

Effects of troglitazone on HepG2 viability and mitochondrial function.

Troglitazone (TRO), a member of the thiazolidinedione class of drugs, has been associated with hepatotoxicity in patients. The following in vitro study was conducted to investigate the effects of TRO on mitochondrial function and viability in a human hepatoma cell line, HepG2. TRO induced a concentration- and time-dependent increase in cell death, as measured by lactate dehydrogenase release. Exposure to 50 or 100 micro M TRO produced total loss of cell viability within 5 h. Preincubation of HepG2 cells with P450 inhibitors did not significantly protect against TRO-induced cell death suggesting that P450 metabolism was not required to induce cell death. Preincubation with the mitochondrial permeability transition inhibitor, cyclosporin A, provided complete protection against TRO-induced cell death. Our results also indicated that TRO produced concentration-dependent decreases in cellular ATP levels and mitochondrial membrane potential (MMP). Ultrastructural analysis demonstrated that TRO induced mitochondrial changes at concentrations of > or =10 micro M after 2 h. Decreased MMP and altered mitochondrial morphology occurred at time points that preceded cell death and at sublethal concentrations of TRO. These observations in HepG2 cells suggest that TRO disrupts mitochondrial function, leading to mitochondrial permeability transition and cell death.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.

Carcinogenicity risk assessment supports the chronic safety of dapagliflozin, an inhibitor of sodium-glucose co-transporter 2, in the treatment of type 2 diabetes mellitus.

INTRODUCTION: Dapagliflozin is a selective inhibitor of the sodium-glucose co-transporter 2 (SGLT2) that increases urinary glucose excretion to reduce hyperglycemia in the treatment of type 2 diabetes mellitus. A robust carcinogenicity risk assessment was undertaken to assess the chronic safety of dapagliflozin and SGLT2 inhibition. METHODS: Genotoxicity potential of dapagliflozin and its metabolites was assessed in silico, in vitro, and in vivo. Dapagliflozin was administered daily by oral gavage to mice, rats, and dogs to evaluate carcinogenicity risks, including the potential for tumor promotion. SGLT2(-/-) mice were observed to evaluate the effects of chronic glucosuria. The effects of dapagliflozin and increased glucose levels on a panel of human bladder transitional cell carcinoma (TCC) cell lines were also evaluated in vitro and in an in vivo xenograft model. RESULTS: Dapagliflozin and its metabolites were not genotoxic. In CD-1 mice and Sprague-Dawley rats treated for up to 2 years at ≥100× human clinical exposures, dapagliflozin showed no differences versus controls for tumor incidence, time to onset for background tumors, or urinary bladder proliferative/preneoplastic lesions. No tumors or preneoplastic lesions were observed in dogs over 1 year at >3,000× the clinical exposure of dapagliflozin or in SGLT2(-/-) mice observed over 15 months. Transcription profiling in Zucker diabetic fatty rats showed that 5-week dapagliflozin treatment did not induce tumor promoter-associated or cell proliferation genes. Increasing concentrations of glucose, dapagliflozin, or its primary metabolite, dapagliflozin 3-O-glucuronide, did not affect in vitro TCC proliferation rates and dapagliflozin did not enhance tumor growth in nude mice heterotopically implanted with human bladder TCC cell lines. CONCLUSION: A multitude of assessments of tumorigenicity risk consistently showed no effects, suggesting that selective SGLT2 inhibition and, specifically, dapagliflozin are predicted to not be associated with increased cancer risk.

Targeting the kidney and glucose excretion with dapagliflozin: preclinical and clinical evidence for SGLT2 inhibition as a new option for treatment of type 2 diabetes mellitus.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a novel class of glucuretic, antihyperglycemic drugs that target the process of renal glucose reabsorption and induce glucuresis independently of insulin secretion or action. In patients with type 2 diabetes mellitus, SGLT2 inhibitors have been found to consistently reduce measures of hyperglycemia, including hemoglobin A1c, fasting plasma glucose, and postprandial glucose, throughout the continuum of disease. By inducing the renal excretion of glucose and its associated calories, SGLT2 inhibitors reduce weight and have the potential to be disease modifying by addressing the caloric excess that is believed to be one of the root causes of type 2 diabetes mellitus. Additional benefits, including the possibility for combination with insulin-dependent antihyperglycemic drugs, a low potential for hypoglycemia, and the ability to reduce blood pressure, were anticipated from the novel mechanism of action and have been demonstrated in clinical studies. Mechanism-related risks include an increased incidence of urinary tract and genital infections and the possibility of over-diuresis in volume-sensitive patients. Taken together, the results of Phase III clinical studies generally point to a positive benefit-risk ratio across the continuum of diabetes patients. To date, data on dapagliflozin, a selective SGLT2 inhibitor in development, demonstrate that the kidney is an efficacious and safe target for therapy, and that SGLT2 inhibition may have benefits for patients with type 2 diabetes mellitus beyond glycemic control.

Targeting renal glucose reabsorption for the treatment of type 2 diabetes mellitus using the SGLT2 inhibitor dapagliflozin.

Sodium-glucose co-transporter 2 (SGLT2) plays a key role in glucose homeostasis as the key transporter responsible for most renal glucose reabsorption in the proximal tubules of the kidney. Dapagliflozin is a potent, selective, and reversible inhibitor of SGLT2 that lowers blood glucose levels in an insulin-independent fashion. This novel agent has been studied extensively in patients with type 2 diabetes mellitus (T2DM). In these clinical trials, dapagliflozin significantly decreased glycated hemoglobin and fasting plasma glucose levels when administered alone or as add-on treatment in patients who were already receiving metformin, a sulfonylurea (glimepiride), pioglitazone, or insulin. Moreover, dapagliflozin decreased body weight when taken as monotherapy or in combination with metformin, a sulfonylurea, or insulin, and mitigated weight gain in patients receiving pioglitazone. Consistent with preclinical toxicology studies, dapagliflozin has a manageable adverse event profile that is largely predictable from its mechanism of action. While there are no clinically significant negative effects on renal function or electrolytes, dapagliflozin treatment is associated with increased frequencies of urinary tract infections and vulvovaginitis/balanitis. With a mechanism of action that is distinct from and complementary to that of existing antihyperglycemic therapies, dapagliflozin is an effective antihyperglycemic agent that is well tolerated and may enhance weight loss. As such, dapagliflozin promises to become an important adjunctive therapy for comprehensive treatment of T2DM.

Cannabinoid receptor antagonist-induced striated muscle toxicity and ethylmalonic-adipic aciduria in beagle dogs.

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.

Validated LC-MS/MS methods for the determination of dapagliflozin, a sodium-glucose co-transporter 2 inhibitor in normal and ZDF rat plasma.

BACKGROUND: Dapagliflozin is an inhibitor of sodium-glucose co-transporter 2 (SGLT-2) in development for the treatment of Type 2 diabetes. To support toxicology studies, LC-MS/MS methods were developed and validated for the quantitation of dapagliflozin in rat plasma. RESULTS: The assay uses solid phase extraction and LC-MS/MS analysis in negative ion electrospray ionization mode. Because dapagliflozin readily forms adducts in the presence of formic acid, the mobile phases were simple mixtures of water and acetonitrile. The assay was validated in the concentration range of 5-2000 ng/ml with good intra- and inter-day precisions and acceptable sample stability. CONCLUSION: The validated assay was successfully applied to the quantitation of dapagliflozin in plasma in support of preclinical studies in both normal and diabetic rats.